PROJECT SUMMARY Ovarian aging is associated with fibrosis, a changing female hormone profile, and a decline in oocyte quality and quantity, resulting in sequelae such as cardiovascular disease, osteoporosis, and infertility. Even before menopause, there are significant age-related changes in hormone production and oocyte quality. Ovarian aging is a fibrotic process involving dramatic extracellular matrix remodeling, resulting in an increasingly rigid microenvironment. While it is known that ECM-derived signals regulate steroidogenesis, the causative relationship between matrix mechanics and function has not previously been demonstrated. Thus, in the studies proposed herein, I will test the hypothesis that mechanical changes in the follicle microenvironment cause a progressive decline in hormone production and egg quality in an aging mouse model. In preliminary studies, we have demonstrated that 3D-printed gelatin scaffolds ? which can be experimentally tuned to different rigidities? support ovarian follicle survival, growth, and function. In Aim 1, I will experimentally manipulate the stiffness of the 3D printed gelatin scaffolds in order to define the effects of rigidity on follicle structure and function and oocyte quality. Specifically, I hypothesize that I can phenocopy older follicles (i.e. diminished hormone production and oocyte quality) by culturing follicles isolated from younger mice in more rigid scaffolds. Conversely, I will perform a rescue experiment with follicles from older animals cultured in a softer scaffold. In Aim 2, proposed experiments will test a possible mechanism of follicle mechanotransduction. In many cell types, Rho/ROCK signaling is responsible for converting mechanical cues into biological response. Moreover, in preliminary studies, we demonstrate that Rho, phospho-Rho, and ROCK are present in discrete follicles in the murine ovary, indicating that Rho signaling is an available molecular mechanism for follicle mechanotransduction. I will assay Rho/ROCK signaling in reproductively younger and older mouse cohorts. Additionally, I will test Rho/ROCK signaling in follicles cultured in 3D printed scaffolds of various rigidities and in the presence of pathway inhibitors. These studies will test my overarching hypothesis that the mechanical properties of the ovarian matrix play a role in age-related ovarian dysfunction (decline in hormone production and egg quality), possibly via mechanosensitive Rho/ROCK signaling. Moreover, these mechanically-tunable 3D-printed scaffolds represent novel in vitro models of ovarian aging and provide a platform for drug discovery and development that may revolutionize the treatment of age-related female infertility.